GmWRKY40, a member of the WRKY transcription factor genes identified from Glycine max L., enhanced the resistance to Phytophthora sojae

Abstract

Background: The WRKY proteins are a superfamily of transcription factors and members play essential roles in the modulation of diverse physiological processes, such as growth, development, senescence and response to biotic and abiotic stresses. However, the biological roles of the majority of the WRKY family members remains poorly understood in soybean relative to the research progress in model plants.

Results: In this study, we identified and characterized GmWRKY40, which is a group IIc WRKY gene. Transient expression analysis revealed that the GmWRKY40 protein is located in the nucleus of plant cells. Expression of GmWRKY40 was strongly induced in soybean following infection with Phytophthora sojae, or treatment with methyl jasmonate, ethylene, salicylic acid, and abscisic acid. Furthermore, soybean hairy roots silencing GmWRKY40 enhanced susceptibility to P. sojae infection compared with empty vector transgenic roots. Moreover, suppression of GmWRKY40 decreased the accumulation of reactive oxygen species (ROS) and modified the expression of several oxidation-related genes. Yeast two-hybrid experiment combined with RNA-seq analysis showed that GmWRKY40 interacted with 8 JAZ proteins with or without the WRKY domain or zinc-finger domain of GmWRKY40, suggesting there were different interaction patterns among these interacted proteins.

Conclusions: Collectively, these results suggests that GmWRKY40 functions as a positive regulator in soybean plants response to P. sojae through modulating hydrogen peroxide accumulation and JA signaling pathway.

Keywords: Glycine max (L.) Merr; GmWRKY40; Phytophthora sojae; RNA interference; Soybean hairy roots; Yeast two-hybrid.

Fig. 1

Characterization and sequence analysis of GmWRKY40. Sequence alignment of the deduced GmWRKY40 protein with Glycine sojae WRKY13 (KHN34020.1), Cajanus cajan WRKY13 (XP_020215926.1), Vigna angularis WRKY 13 (XP_017432586.1), Nicotiana tabacum WRKY 28 (XP_016452899.1) and Arabidopsis thaliana WRKY13 (NP_195651.1). The dark blue (100%), pink (80%), and cyan (60%) boxes represent levels of amino acid identity or similarity. The double arrow indicates the WRKY domain. The highly conserved amino acid sequence is boxed. The C and H residues in the zinc-finger motif are marked by red dots

Fig. 2

Subcellular localization of the GmWRKY40 protein. a Schematic diagram of the 35S-GmWRKY40::GFP fusion construct and the 35S-GFP construct. b Agrobacteria carrying 35S-GmWRKY40::GFP and 35S-GFP were infiltrated into leaves of N. benthamiana plants. Pictures were taken in bright and fluorescence fields after DAPI staining with confocal laser scanning microscopy 72 h after agroinfiltration

Fig. 3

Expression patterns of GmWRKY40 under diverse stress conditions. Approximately 1-week-old soybean seedlings were used for each of the following treatments. a The relative expression of GmWRKY40 was determined at different time points in the soybean hypocotyl after inoculation with P. sojae. b-e GmWRKY40 transcripts examined in soybean roots at various time periods after treatment with 100 μM MeJA, 2 mM ETH, 5 mM SA and 50 mM ABA. Error bars indicate the standard error; the experiments were repeated three times along with at least three independent repetitions of the biological experiments. Asterisks indicate statistically significant differences (^**P < 0.01)

Fig. 4

Transgenic soybean hairy roots with silenced GmWRKY40 exhibit enhanced susceptibility to Phytophthora sojae. a Disease phenotypes of GmWRKY40-RNAi Williams 82 hairy roots and control at 36 h after inoculated with P6497. b The lesion length of P6497 infected hairy roots. Lesion length was taken 36 hpi. c Relative expression of GmWRKY40 was determined by qPCR at 12 and 24 hpi in GmWRKY40 silencing or EV hairy roots (d) P. sojae biomass was determined by qPCR at 12 and 24 hpi in GmWRKY40 silencing or EV hairy roots. The experiments all above were repeated three times along with at least three independent repetitions of the biological experiments. Error bars indicate the standard error. Asterisks indicate statistically significant differences (^**P < 0.01)

Fig. 5

GmWRKY40 regulates soybean defense against P. sojae infection. a GmWRKY40 silenced hairy roots were hyper-susceptible to P. sojae infection. Hairy roots were inoculated with zoospore suspension (10⁴ zoospore/mL) of the P. sojae P6497R. P. sojae infection was monitored using a fluorescent microscope at 24, 36 and 48 hpi. Bar equals 0.5 mm. b Infection progress of P. sojae in soybean roots expressing EV or RNAi-GmWRKY40. The number of roots allowing hyphae penetration or oospore development were counted at 24, 36 and 48 hpi. This experiment were repeated three times with similar results

Fig. 6

Silencing GmWRKY40 decreased the accumulation of ROS following P. sojae infection. a Accumulation of H₂O₂ was detected by DAB staining at 6, 12 and 24 h after inoculated with P. sojae. This experiment was repeated three times with similar results. b H₂O₂ content was measured at 6, 12 and 24 h after inoculated with P. sojae. Values represent the means of 3 replicates ± SD. Asterisks indicate statistically significant differences (*P < 0.05 or **P < 0.01). c-e Transgenic hairy roots were inoculated with zoospore suspension of P. sojae and samples were collected at 0 or 24 h after inoculation. Gene expression of GmNADPHox, GmAPX1 and GmCAT1 were analyzed by qRT-PCR. Values represent the means of 3 replicates ± SD. ^** Student’s t-test: p < 0.01

Fig. 7

GmWRKY40 interacts with JAZ proteins. a Diagram of the GmWRKY40 protein functional domains and schematic of the amino acid mutation of WRKYGQK domain or Zinc finger domain. mWRKY indicate WRKY domain mutation; mZinc-finger indicate Zinc-finger domain mutation. b Yeast-two-hybrid assays. The Gal4 DNA binding domain was fused with GmWRKY40 (shown as BD-WRKY40, BD-mWRKY or BD-mZinc-finger) and the Gal4 activation domain was fused with JAZ1, JAZ2 or JAZ3 (shown as AD-JAZ1, AD-JAZ2 or AD-JAZ3). The Gal4 DNA binding domain expressed by pGBKT7 was used as a negative control. 0.8, 0.08, and 0.008 are the bacteria diluted concentration gradient

Fig. 8

GmWRKY40 interacts with multiple JAZ family proteins. Yeast-two-hybrid assays. The Gal4 DNA binding domain was fused with GmWRKY40 (shown as BD-WRKY40, BD-mWRKY or BD-mZinc-finger) and the Gal4 activation domain was fused with JAZ4-JAZ8 (shown as AD-JAZ4 to AD-JAZ8). The Gal4 DNA binding domain expressed by pGBKT7 was used as a negative control. 0.8, 0.08, and 0.008 are the bacteria diluted concentration gradient